please help me check BSP primers. - (Apr/15/2008 )

I have succeeded designing MSP primers,but been having trouble with BSP primers. By using Methprimer,I designed primers.Unfortunately, products are too little to be seen,even run the 2ed pcr with the same primers.It's difficult for me designing nested or hemi-nested primers.Is anyone here who could help check and design nested BSP primer? Thanks a lot in advance! The original and my targeted sequence are pasted below.
gcaggctgtgggatgtgcaccaggttgatacagaacccagctccaccaagctgaaagggcgcgtcgcccgggccagggtg
gggcaccaggttatgagtggcctcctgccttttgggtttgcttccccgcaggggacaccgtaggcggctgtgcggtccggc
cactgccccccgcccctcgcgtccggccgcgccgtgggttgcggtctccgtgggggtggcagctcctgtccgggaggtttg
cgcctggcggggcaccccagagccgcgaagagccggtaggcgaggccagggcggggtggggcgcgtccaggcggggagggc
aaactcgggcagcgcagggggcgcagagggcagcgggcgggcggacgcgcggcggaggcgcgagcgggacgagggctggct
agtgccggggccgcccgcccgagggggaggaggctgtgctgcccttgctgcaggttcgctgtcgagcgcacaggaggcagg
gacatgggtagggtgcggtctgcgcggggcccgagcccggatctcttccatttcccctctcactcggccccgggctgcgcc
gcagacggcagcagctcccgctccgcccgagccgcctgaccgccgggccggggtgctaaccgcggggccctgcagcccgcc
ggcccggccagcccagcccagcccggcggcccgcagccccgccgcccgccgccccccgccgccgccgcgttgccaaaacaaacgggccggcctatttattggcggc
cggcgagcccggcagctcagagtcgaggcgccgagggggacagcgcgccgccaccagctcgggccctgggcccccgccccg
cacttgagtcccgccggccctggccgcaccacgccgcccatggcgcccccgcctggagccccccggagccacccggacgcct
gagcccccgcagcgctcccgtcgacgcgcctgcccatcagcccaccaggagacctcgcccgccgctcccccgggctccccg
gcc
my primers
sense:GTATAGGAGGTAGGGATATGGGTAG
antisense:TCTCCTAATAAACTAATAAACAAAC
Product size: 497, Tm:51-58, CpGs in product: 65
PCR cycling profile.
95°C 5 min
95°C 30 sec
48°C 90 sec
64°C 45 sec
72°C 45 sec (2 ex tension temp)
× 35cycles.

72°C 8minutes
4°C hold.

hi fxs,

I have always been unhappy with the primers picked by methprimer.

I have picked a couple for you, adjust the length of the primer from the 5'end (longer or shorter) so that they have equivalent Tm approx 60C.

note that the primer should end in a conversion event (a T that was a C, in bold)

Good luck

Nick

you should try them out for sequencing,

I have done so in the past and got funny results (lots of non-CpG methylation).

Nick

After 2 rounds PCR with same primers which methprimer designed,it worked. I will try primers Nick designed for nested pcr.
I found that MSP primers from methprimer always work.
But,when I study methylation status of GLI-1 gene of human,I am getting totaly frustrated.I have tried MSP and BSP with 6 couples of primers methprimer designed,it just didn't work through 2 months of hard work .Experts here are very helpful.Anyone could help solve the problem?thanks a lot.
The original and my targeted sequence are pasted below.
ttttatgctccatagactcctcaccttcttccagagcccccaacccaacttgatttgccccaaaccgcaactctgtcccg
gccgctgcaagttccatccaaagggtgaggcctgcagataaaccacaggatggcagaatgctcagttagcaccaaccaaag
gcgactaccctacctccactattatcgttctcggttgaacttctccccctgccccgcaatattttcctcaatctggttgtc
ggggcctctttggggccagccgatccagaaatccaagccgggatttagtactcaccaacagcagcgtgttca gccggggcgggggggggggcgtaagcagtatagggtccctcaagggagggggaggatcctgggggtcctgggggtgcaat
aagcccggcaccccttctcttgcttccagctaccccgcctcatcctccagaacggcaagagggagggaaatagaagggagg
tgaggggcgagcgggaagagcggcggcgcgccagcggctggagagagaaaaagtttttgcaaaagggaaaaaaaaagtttg
cgcttctcgcgggtggtccgggcttgcggcccggcgggctgggccggcgggagggctgggggccaggttgggggggtgggg
gtggcatcgaggctgcgctgccgtggccctctccgccccccctccccaccgcacaccccccagCCCAGACTCCAGCCCTGG
ACCGCGCATCCCGAGCCCAGCGCCCAGACAGAGgtgagaagggggggcaggcgggggaccacctgggagcagtgggggagg
gggcctgagggg
atgctcagcttcttagggactcatcccagacccgggacatagaggcaaaataggggtgggagagcctggggtgagacatt
agaaactccagatttttcacttgtgtctttctctgtatcttctttttcttccctttttttcttctgtcagtctgtgtatct
ctgtctcagggaaccgtgggtctttgtctccgcctctcccatatattagaaatatcttactttcatgcggttaagtttaag
aggctggagggatggctagctggaggtctgcgttgtagagaggtaaccccaggtgtgtgtctgcgcgtggggtaggaagat
gtcagtgtttctgaaaggtggggactgcaaaggagggag

my BSP primers
F1：GGTTTTGGGGGTGTAATAAG
R1：CCCCTCCCCCACTACTCC
424bp

F2：GGAAATAGAAGGGAGGTGAG
R2：CCCCCTTCTCACCTCTATCTA
304bp

F3： GGTTTTGGGGGTGTAATAAGTT
R3： CCCCCTTCTCACCTCTATCTAAA
386bp

MSP primers
M F： 5- AGTTTAGATTTTAGTTTTGGATCGC-3
M R： 5- CCCTATTTTACCTCTATATCCCGAA-3
168bp Tm 72.4
U F：5- GTTTAGATTTTAGTTTTGGATTGTGT-3
U R：5- CCCTATTTTACCTCTATATCCCAAA-3
167bp Tm 70.3

After 2 rounds PCR with same primers which methprimer designed,it worked. I will try primers Nick designed for nested pcr.
I found that MSP primers from methprimer always work.
But,when I study methylation status of GLI-1 gene of human,I am getting totaly frustrated.I have tried MSP and BSP with 6 couples of primers methprimer designed,it just didn't work through 2 months of hard work .Experts here are very helpful.Anyone could help solve the problem?thanks a lot.
The original and my targeted sequence are pasted below.
ttttatgctccatagactcctcaccttcttccagagcccccaacccaacttgatttgccccaaaccgcaactctgtcccg
gccgctgcaagttccatccaaagggtgaggcctgcagataaaccacaggatggcagaatgctcagttagcaccaaccaaag
gcgactaccctacctccactattatcgttctcggttgaacttctccccctgccccgcaatattttcctcaatctggttgtc
ggggcctctttggggccagccgatccagaaatccaagccgggatttagtactcaccaacagcagcgtgttca gccggggcgggggggggggcgtaagcagtatagggtccctcaagggagggggaggatcctgggggtcctgggggtgcaat
aagcccggcaccccttctcttgcttccagctaccccgcctcatcctccagaacggcaagagggagggaaatagaagggagg
tgaggggcgagcgggaagagcggcggcgcgccagcggctggagagagaaaaagtttttgcaaaagggaaaaaaaaagtttg
cgcttctcgcgggtggtccgggcttgcggcccggcgggctgggccggcgggagggctgggggccaggttgggggggtgggg
gtggcatcgaggctgcgctgccgtggccctctccgccccccctccccaccgcacaccccccagCCCAGACTCCAGCCCTGG
ACCGCGCATCCCGAGCCCAGCGCCCAGACAGAGgtgagaagggggggcaggcgggggaccacctgggagcagtgggggagg
gggcctgagggg
atgctcagcttcttagggactcatcccagacccgggacatagaggcaaaataggggtgggagagcctggggtgagacatt
agaaactccagatttttcacttgtgtctttctctgtatcttctttttcttccctttttttcttctgtcagtctgtgtatct
ctgtctcagggaaccgtgggtctttgtctccgcctctcccatatattagaaatatcttactttcatgcggttaagtttaag
aggctggagggatggctagctggaggtctgcgttgtagagaggtaaccccaggtgtgtgtctgcgcgtggggtaggaagat
gtcagtgtttctgaaaggtggggactgcaaaggagggag

my BSP primers
F1：GGTTTTGGGGGTGTAATAAG
R1：CCCCTCCCCCACTACTCC
424bp

F2：GGAAATAGAAGGGAGGTGAG
R2：CCCCCTTCTCACCTCTATCTA
304bp

F3： GGTTTTGGGGGTGTAATAAGTT
R3： CCCCCTTCTCACCTCTATCTAAA
386bp

MSP primers
M F： 5- AGTTTAGATTTTAGTTTTGGATCGC-3
M R： 5- CCCTATTTTACCTCTATATCCCGAA-3
168bp Tm 72.4
U F：5- GTTTAGATTTTAGTTTTGGATTGTGT-3
U R：5- CCCTATTTTACCTCTATATCCCAAA-3
167bp Tm 70.3

Hi,larryking.Thanks a lot.
Recently,my RT-PCR found the gene doesn't express differently among groups. It now seems studying the methylation of the gene meaningless. I have turned to other gene.